Microorganisms for producing putrescine and process for producing putrescine using them

ABSTRACT

Disclosed is a modified microorganism producing putrescine or ornithine, and a method for producing putrescine or ornithine using the same.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/746,300, filed on Jan. 19, 2018, which is a National Stageapplication under 35 U.S.C. § 371 of International Application No.PCT/KR2016/007841, filed Jul. 19, 2016, which claims the benefit under35 U.S.C. § 119(a) of Korean Patent Application No. 10-2015-0102624,filed Jul. 20, 2015, the contents of all of which are incorporatedherein in their entireties by reference thereto.

SEQUENCE LISTING

This application contains a Sequence Listing which has been submitted inASCII format via EFS and is hereby incorporated by reference. The ASCIIcopy, created on Jan. 16, 2018, and amended on Apr. 16, 2018, is namedOPA16079_Seq_List.txt and is 89,023 bytes in size.

TECHNICAL FIELD

The present disclosure relates to a recombinant microorganism producingputrescine or ornithine, and a method for producing putrescine orornithine using the same.

BACKGROUND ART

Biogenic amines (BAs) are nitrogen compounds that are mainly produced bydecarboxylation of amino acids or amination and transamination ofaldehydes and ketones. These biogenic amines have low molecular weightand are synthesized during the metabolic processes in microorganisms,plants, and animals thus being known as constituting elements which arefrequently discovered in their cells.

Among them, putrescine is discovered in gram negative bacteria or fungiand is present in high concentration in various species, and thusputrescine is expected to play an important role in the metabolism ofmicroorganisms. In general, putrescine is an important raw material forthe synthesis of polyamine nylon-4,6 and is produced mainly by chemicalsynthesis. The chemical synthesis is a 3-step process including acatalytic oxidation reaction, a reaction using a cyanide compound, and ahydrogenation reaction using high-pressure hydrogen. Accordingly, thereis a demand for the development of a more environment-friendly andenergy-effective method involving biomass utilization.

Under these circumstances, various methods for producing putrescine athigh concentration by transforming E. coli and a microorganism of thegenus Corynebacterium were disclosed (International Patent PublicationNo. WO 06/005603; International Patent Publication No. WO 09/125924;Qian Z D et al., Biotechnol. Bioeng. 104 (4): 651-662, 2009; Schneideret al., Appl. Microbiol. Biotechnol. 88 (4): 859-868, 2010; Schneider etal., Appl. Microbiol. Biotechnol. 95: 169-178, 2012).

On the other hand, ornithine is a material widely discovered in plants,animals, and microorganisms, and serves as a precursor for biosynthesisof arginine, proline, and polyamine. Additionally, ornithine plays animportant role in the pathway of producing urea from amino acids orammonia and disposing through the ornithine cycle during the in-vivometabolism of higher organisms. Ornithine is effective in muscleproduction and reduction of body fat, and thus it has been used as anutrient supplement and also as a pharmaceutical drug for improvingliver cirrhosis and hepatic dysfunction. Methods of producing ornithineinclude a method of using milk casein as a digestive enzyme and a methodof using E. coli or a microorganism of the genus Corynebacterium (KoreanPatent No. 10-1372635; T. Gotoh et al., Bioprocess Biosyst. Eng., 33:773-777, 2010).

E. coli and a microorganism of the genus Corynebacterium are similar inthe biosynthetic pathways for producing putrescine or ornithine, butthey also exhibit differences as follows. First, the microorganism ofthe genus Corynebacterium has a “cyclic pathway”, in which glutamic acidis converted into N-acetyl-L-glutamic acid and N-acetyl-L-ornithine isconverted into L-ornithine by argJ (bifunctional ornithineacetyltransferase/N-acetylglutamate synthase, EC 2.3.1.35). In contrast,E. coli is involved in the biosynthesis of putrescine or ornithine by a“linear pathway”, in which argA (N-acetylglutamate synthase, EC 2.3.1.1)and argE (Acetylornithine deacetylase, EC 3.5.1.16) replace the role ofthe argJ in the microorganism of the genus Corynebacterium.

In the microorganism of the genus Corynebacterium, it is known that anacetyl group recycles between omithine and glutamic acid in ArgJ.However, in E. coli, ArgA attaches the acetyl group of acetyl-CoA toglutamate in order to produce N-acetylglutamate, and ArgEN-acetyl-omithine decomposes N-acetyl-omithine to produce omithine andacetate (Schneider et al., Appl. Microbiol. Biotechnol. 91, 17-30,2011).

In particular, pta-ackA (pta, phosphotransacetylase; ackA, acetatekinase) operon and acetyl-coenzyme A synthetase (acs) are known as genesto synthesize acetyl-CoA using acetate.

DISCLOSURE Technical Problem

The present inventors have made many efforts to improve the ability of amicroorganism of the genus Corynebacterium to produce ornithine andputrescine, and as a result they have discovered that the introductionof E. coli-derived argA and argE into a microorganism of the genusCorynebacterium can improve its ability to produce ornithine andputrescine, thereby completing the present invention.

Technical Solution

An object of the present disclosure provides a recombinant microorganismproducing putrescine or ornithine in high yield.

Another object of the present disclosure provides a method for producingputrescine or omithine using the microorganism above.

Advantageous Effects of the Invention

It was confirmed that the microorganism of the genus Corynebacterium ofthe present disclosure producing putrescine or omithine can improve theamount of putrescine- or omithine production when the microorganism isintroduced with E. coli-derived argA and E. coli-derived argE, and alsowhen the acetate utilization pathway is reinforced. Accordingly, themicroorganism of the present disclosure can be widely used for theproduction of putrescine or omithine, and also, can be widely used as aneffective and desirable means to supply raw materials for the productionof various polymer products, in which the putrescine or ornithine isused as a raw material, from the economic and environmental aspects.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic diagram illustrating a biosynthetic pathway (acyclic pathway) for producing putrescine and ornithine in amicroorganism of the genus Corynebacterium (A) and a biosyntheticpathway (a linear pathway) for producing putrescine and ornithine in E.coli (B).

FIG. 2 is a schematic diagram illustrating the biosynthetic pathwaywhich has improved the ability to produce putrescine and ornithine byintroducing E. coli-derived argA and E. coli-derived argE into amicroorganism of the genus Corynebacterium, which is in a stateexpressing argJ.

BEST MODE

An aspect of the present disclosure provides a modified microorganism ofthe genus Corynebacterium producing putrescine or ornithine, in whichactivities of N-acetylglutamate synthase from E. coli andacetylornithine deacetylase from E. coli are introduced.

An exemplary embodiment of the present disclosure provides a modifiedmicroorganism of the genus Corynebacterium producing putrescine orornithine, in which the E. coli-derived N-acetylglutamate synthaseconsists of an amino acid sequence of SEQ ID NO: 1.

Another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor ornithine, in which the E. coli-derived acetylornithine deacetylaseconsists of an amino acid sequence of SEQ ID NO: 3.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor ornithine, in which the microorganism of the genus Corynebacterium isselected from the group consisting of Corynebacterium glutamicum,Corynebacterium ammoniagenes, Corynebacterium thermoaminogenes,Brevibacterium flavum, and Brevibacterium lactofermentum.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of phosphotransacetylase and acetatekinase operon (pta-ackA operon) is further enhanced compared to itsendogenous activity.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which the phosphotransacetylase and acetate kinaseoperon consists of an amino acid sequence of SEQ ID NO: 5 or 7.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of E. coli-derived acetyl-CoAsynthetase (acs) is further introduced.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which the E. coli-derived acetyl-CoA synthetase (acs)consists of an amino acid sequence of SEQ ID NO: 9.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of ornithine decarboxylase (ODC) isfurther introduced.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of i) omithine carbamoyltransferase(ArgF), ii) glutamate exporter, or iii) omithine carbamoyltransferaseand glutamate exporter is further weakened, compared to its endogenousactivity.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of at least one selected from thegroup consisting of acetyl gamma glutamyl phosphate reductase (ArgC),acetylglutamate synthase or omithine acetyltransferase (ArgJ),acetylglutamate kinase (ArgB), and acetyl ornithine aminotransferase(ArgD), is further enhanced compared to its endogenous activity.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of acetyltransferase is furtherweakened compared to its endogenous activity.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which the acetyltransferase consists of the amino acidsequence of SEQ ID NO: 30 or 31.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which an activity of the putrescine exporter is furtherenhanced compared to its endogenous activity.

Still another exemplary embodiment of the present disclosure provides amodified microorganism of the genus Corynebacterium producing putrescineor omithine, in which the putrescine exporter consists of the amino acidsequence of SEQ ID NO: 26 or 28.

Another aspect of the present disclosure provides a method for producingputrescine or omithine, including:

(i) culturing the modified microorganism of the genus Corynebacteriumproducing putrescine or omithine in a medium; and

(ii) recovering putrescine or ornithine from the cultured microorganismor the medium.

In an exemplary embodiment of the present disclosure, the modifiedmicroorganism of the genus Corynebacterium is Corynebacteriumglutamicum.

Hereinafter, the present disclosure will be described in detail.

An embodiment of the present disclosure provides a modifiedmicroorganism of the genus Corynebacterium producing putrescine orornithine, in which activities of E. coli-derived N-acetylglutamatesynthase and E. coli-derived acetylomithine deacetylase are introduced.

As used herein, the term “N-acetylglutamate synthase” refers to anenzyme which mediates the reaction producing N-acetylglutamate fromglutamate and acetyl-CoA, and the N-acetylglutamate produced thereof maybe used as a precursor of ornithine and arginine.

In the present disclosure, N-acetylglutamate synthase may include, forexample, the protein having an amino acid sequence of SEQ ID NO: 1, andany protein, which has a homology of 70% or higher, specifically 80% orhigher, more specifically 90% or higher, even more specifically 95% orhigher, yet even more specifically 98% or higher, and most specifically99% or higher, to the amino acid sequence above, as long as the proteinhas the substantial activity of N-acetylglutamate synthase, withoutlimitation.

Additionally, the proteins exhibiting the activity above may showdifferences in amino acid sequences, according to the species andstrains of the microorganism. Accordingly, the N-acetylglutamatesynthase of the present disclosure may be, for example, one from E.coli, although it is not limited thereto.

As a sequence having a homology to the sequence above, if the amino acidsequence is one which has substantially the same or corresponding tobiological activity of a protein of SEQ ID NO: 1, it is obvious in thatamino acid sequences with a deletion, a modification, a substitution, oran addition in part of the sequences should also be included in thescope of the present disclosure.

The polynucleotide encoding the N-acetylglutamate synthase of thepresent disclosure may include, without limitation, a polynucleotideencoding the protein having an amino acid sequence of SEQ ID NO: 1, andany protein, which has a homology of 70% or higher, specifically 80% orhigher, more specifically 90% or higher, even more specifically 95% orhigher, yet even more specifically 98% or higher, and most specifically99% or higher, to the above amino acid sequence, as long as thepolynucleotide has an activity similar to that of N-acetylglutamatesynthase, and for example, a polynucleotide sequence of SEQ ID NO: 2 maybe included.

As used herein, the term “acetylornithine deacetylase” refers to anenzyme which mediates the reaction involved in the production of aceticacid and ornithine by mediating the hydrolysis of acetylornithine.

In the present disclosure, acetylornithine deacetylase may include,without limitation, the protein having an amino acid sequence of SEQ IDNO: 3, and any protein, which has a homology of 70% or higher,specifically 80% or higher, more specifically 90% or higher, even morespecifically 95% or higher, yet even more specifically 98% or higher,and most specifically 99% or higher, to the above amino acid sequence,as long as the protein has the substantial activity of separating acetylgroup and ornithine from acetylornithine.

Additionally, the proteins exhibiting the activity above may show adifference in amino acid sequences, according to the species and strainsof the microorganism. Accordingly, the acetylornithine deacetylase ofthe present disclosure may be one from E. coli, although it is notlimited thereto. As a sequence having a homology, if the amino acidsequence is one which has substantially the same or corresponding tobiological activity of a protein of SEQ ID NO: 3, it is obvious in thatamino acid sequences with a deletion, a modification, a substitution, oran addition in part of the sequences should also be included in thescope of the present disclosure.

The polynucleotide encoding acetylornithine deacetylase of the presentdisclosure may include, as long as the polynucleotide has an activitysimilar to that of the acetylornithine deacetylase protein, the proteinhaving an amino acid sequence of SEQ ID NO: 3, or a polynucleotideencoding a protein, which has a homology of 70% or higher, specifically80% or higher, more specifically 90% or higher, even more specifically95% or higher, yet even more specifically 98% or higher, and mostspecifically 99% or higher, to the amino acid sequence above, forexample, a polynucleotide sequence of SEQ ID NO: 4.

Additionally, the polynucleotide encoding N-acetylglutamate synthase oracetylornithine deacetylase of the present disclosure may be hybridizedwith the polynucleotide sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or aprobe derived from the polynucleotide sequence under stringentconditions, and it may be a modified type of N-acetylglutamate synthaseor acetylornithine deacetylase that functions normally. In the above,the term “stringent conditions” refers to a condition that enables aspecific hybridization between polynucleotides. For example, thestringent conditions are specifically described in references (e.g., J.Sambrook et al., supra).

In the above, the term “homology” refers to the degree of identity withthe given amino acid sequence or a polynucleotide sequence, and may beindicated in percentage. As used herein, the homologous sequence havingthe same or similar activity with the given polypeptide sequence orpolynucleotide sequence may be indicated in terms of “% homology”. Forexample, the % homology may be confirmed using standard software, i.e.,BLAST 2.0, for calculating parameters such as score, identity, andsimilarity, or by comparing sequences via southern hybridizationexperiments, and the appropriate hybridization condition to be definedmay be determined by a method known to a skilled person in the art(e.g., J. Sambrook et al., Molecular Cloning, A Laboratory Manual, 2ndEdition, Cold Spring Harbor Laboratory press, Cold Spring Harbor, N.Y.,1989; F. M. Ausubel et al., Current Protocols in Molecular Biology, JohnWiley & Sons, Inc., New York).

On the other hand, the microorganism of the present disclosure mayinclude both a natural type and a modified type, e.g., microorganismsthat belong to the genus Escherichia, the genus Shigella, the genusCitrobacter, the genus Salmonella, the genus Enterobacter, the genusYersinia, the genus Klebsiella, the genus Erwinia, the genusCorynebacterium, the genus Brevibacterium, the genus Lactobacillus, thegenus Selenomanas, the genus Vibrio, the genus Pseudomonas, the genusStreptomyces, the genus Arcanobacterium, the genus Alcaligenes, etc.Specifically, the microorganism of the present disclosure may be amicroorganism belonging to the genus Corynebacterium, more specifically,a microorganism selected from the group consisting of Corynebacteriumglutamicum, Corynebacterium ammoniagenes, Corynebacteriumthermoaminogenes, Brevibacterium flavum, and Brevibacteriumlactofermentum, and more specifically, Corynebacterium glutamicum, butit is not limited thereto.

Specifically, as used herein, the term “a microorganism of the genusCorynebacterium producing putrescine or ornithine” refers to amicroorganism of the genus Corynebacterium producing putrescine orornithine in a natural state; or a microorganism of the genusCorynebacterium producing putrescine or ornithine prepared by providingthe ability to produce putrescine or ornithine into its parent strain,which cannot produce putrescine or ornithine.

The microorganism, which is provided with the ability to produceputrescine or ornithine or can produce putrescine or ornithine, may havean improved ability to produce ornithine, which is used as a rawmaterial for biosynthesis of putrescine, by modifying the activities ofacetylglutamate synthase (which converts glutamate intoN-acetylglutamate), ornithine acetyltransferase (ArgJ, which convertsacetylornithine into ornithine), acetylglutamate kinase (ArgB, whichconverts acetylglutamate into N-acetylglutamyl phosphate), gammaglutamyl phosphate reductase (ArgC, which converts N-acetylglutamylphosphate into N-acetylglutamate semialdehyde), and acetyl ornithineaminotransferase (ArgD, which converts acetylglutamate semialdehyde intoN-acetylornithine) to be increased, compared to their endogenousactivities, in order to increase the biosynthetic pathway from glutamateto ornithine, although not particularly limited thereto.

Additionally, the microorganism may be modified to weaken the activitiesof ornithine carbamoyltransferase (ArgF, which is involved in thesynthesis arginine from ornithine), a protein(s) involved in the exportof glutamate, and/or a protein(s) that acetylates putrescine, comparedto their endogenous activities; and/or to introduce the activity ofornithine decarboxylase (ODC).

As used herein, the term “introduction of activity” may refer to anactivity of a protein, which is not present or weak in a microorganism,is newly introduced or enhanced in the corresponding microorganism.Specifically, it may include inserting or delivering a gene encoding aprotein, which is not present in the microorganism, into themicroorganism to be expressed therein, or inducing a modification of theprotein for enhancing the expression of the protein, which is notexpressed or almost not expressed in the microorganism, but is notlimited thereto.

On the other hand, in the present disclosure, modifications such asintroduction of activity, enhancement of activity, weakening ofactivity, etc., may occur through a process called transformation. Asused herein, the term “transformation” refers to a process ofintroducing a vector, which includes a polynucleotide encoding aparticular protein or a promoter sequence with strong or weak activity,etc., into the host cell thereby enabling the expression of the proteinencoded by the polynucleotide or inducing a modification of thechromosome in the host cell. Additionally, the polynucleotide includesDNA and RNA which encode the target protein. The polynucleotide may beinserted in any form insofar as it can be introduced into a host celland expressed or induce a modification therein. For example, thepolynucleotide may be introduced into a host cell in the form of anexpression cassette, which is a gene construct including all essentialelements required for self-expression. The expression cassette mayconventionally include a promoter operably connected to thepolynucleotide, a transcription termination signal, a ribosome-bindingdomain, and a translation termination signal, and may be in the form ofan expression vector capable of self-replication. Additionally, thepolynucleotide may be introduced into a host cell as it is, and operablyconnected to a sequence essential for its expression in the host cell,but is not limited thereto.

Additionally, as used herein, the term “operably connected” refers to afunctional connection between a promoter sequence, which initiates andmediates the transcription of the polynucleotide encoding the particularprotein of the present disclosure, and the gene sequence.

As used herein, the term “vector” refers to a DNA construct includingthe nucleotide sequence of the polynucleotide encoding a protein ofinterest, in which the protein of interest is operably linked to asuitable regulatory sequence so that the protein of interest can beexpressed in an appropriate host. The regulatory sequence includes apromoter capable of initiating transcription, any operator sequence forregulation of the transcription, a sequence encoding a suitable mRNAribosome-binding domain, and a sequence for regulating transcription andtranslation. The vector, after being transformed into a suitable hostcell, may be replicated or function irrespective of the host genome, ormay be integrated into the host genome itself.

The vector used in the present disclosure may not be particularlylimited as long as the vector is replicable in a host cell, and anyvector known in the art may be used. Examples of the vector may includenatural or recombinant plasmids, cosmids, viruses, and bacteriophages.For example, as a phage vector or cosmid vector, pWE15, M13, MBL3, MBL4,IXII, ASHII, APII, t10, t11, Charon4A, Charon21A, etc., may be used; andas a plasmid vector, those based on pBR, pUC, pBluescriptII, pGEM, pTZ,pCL, pET, etc., may be used. The vector to be used in the presentdisclosure may not be particularly limited and any vector known in theart may be used. Specifically, pDZTn, pACYC177, pACYC184, pCL, pECCG117,pUC19, pBR322, pMW118, pCC1BAC vectors, etc., may be used.

As such, a polynucleotide encoding a target protein may be substitutedwith a modified polynucleotide using a vector for chromosomal insertionwithin bacteria. The insertion of the polynucleotide into the chromosomemay be performed using a known method in the art, for example, byhomologous recombination, but is not limited thereto. Since the vectorof the present disclosure can be inserted into the chromosome viahomologous recombination, a selection marker for confirming theinsertion into the chromosome may be further included. The selectionmarker is used for selecting a transformed cell, i.e., in order toconfirm whether the target polynucleotide has been inserted, and markersproviding selectable phenotypes such as drug resistance, nutrientrequirement, resistance to cytotoxic agents, and expression of surfaceproteins may be used. Under the circumstances where selective agents aretreated, only the cells expressing the selection markers can survive orexpress other phenotypic traits, and thus the transformed cells can beeasily selected.

The microorganism of the genus Corynebacterium of the present disclosuremay be a modified microorganism of the genus Corynebacterium producingputrescine or ornithine, in which an activity of phosphotransacetylaseand acetate kinase operon (pta-ackA operon) is further enhanced comparedto its endogenous enzyme.

In the present disclosure, the phosphotransacetylase and acetate kinaseoperon (pta-ackA operon) are operons including genes that reversiblymediate the metabolic pathway, in which acetyl-CoA produced from glucoseor pyruvate converts into acetic acid via acetyl phosphate, and themetabolic pathway in the opposite direction.

In the present disclosure, the phosphotransacetylase and acetate kinaseoperon may include, without limitation, the proteins including an aminoacid sequence of SEQ ID NO: 5 or SEQ ID NO: 7, or any protein, which hasa homology of 70% or higher, specifically 80% or higher, morespecifically 90% or higher, specifically, even more specifically 95% orhigher, yet even more specifically 98% or higher, or most specifically99% or higher, to the above amino acid sequences, as long as the proteinsubstantially mediates the reaction of producing acetyl-CoA from aceticacid.

Additionally, since the amino acid sequences of the proteins exhibitingthe activities may vary according to the species or strains of a givenmicroorganism, the phosphotransacetylase and acetate kinase operon inthe present disclosure may not be limited to those origins from whichthey are derived. It is obvious in that any amino acid sequence, whichhas a homology to the sequences above and has a biological activitysubstantially the same as or corresponding to the protein of SEQ ID NO:5 or SEQ ID NO: 7, can also belong to the scope of the presentdisclosure, although the amino acid sequence may have deletion,modification, substitution, or addition, in part of the sequence.

The polynucleotide encoding the phosphotransacetylase and acetate kinaseoperon of the present disclosure may include the polynucleotide whichencodes the amino acid of SEQ ID NO: 5 or SEQ ID NO: 7, or thepolynucleotide which encodes a protein having a homology of 70% orhigher, specifically 80% or higher, more specifically 90% or higher,even more specifically 95% or higher, yet even more specifically 98% orhigher, and most specifically 99% or higher to the above amino acidsequences, and most specifically may include the polynucleotide sequenceof SEQ ID NO: 6 or SEQ ID NO: 8.

As used herein, the term “enhancement of activity” not only includes thedrawing of a higher effect than the original function due to the newintroduction of an activity or an increase in the activity of a proteinitself, but also includes the increase in its activity by an increase inthe activity of an endogenous gene, amplification of an endogenous genefrom internal or external factor(s), deletion of regulatory factor(s)for inhibiting the gene expression, an increase in gene copy number,introduction of a gene from outside, modification of the expressionregulatory sequence, and specifically, an increase in enzyme activitydue to replacement or modification of a promoter and a mutation withinthe gene, etc.

Specifically, in the present disclosure, the increase in activity may beperformed by:

-   -   1) increasing copy number of a polynucleotide encoding the        enzyme,    -   2) modifying the expression regulatory sequence for increasing        the expression of the polynucleotide,    -   3) modifying the polynucleotide sequence on the chromosome for        enhancing the activity of the enzyme, or    -   4) modifying by a combination thereof,        -   but the method is not limited thereto.

The increase of copy number of a polynucleotide (method 1) may beperformed in a form in which the polynucleotide is operably linked to avector, or by inserting the polynucleotide into the chromosome of a hostcell, although the method is not particularly limited thereto.Specifically, the copy number of a polynucleotide within the chromosomeof the host cell may be increased by introducing a vector which canreplicate and function regardless of a host cell and to which thepolynucleotide encoding the protein of the present disclosure isoperably linked; or may be increased by introducing a vector, which caninsert the polynucleotide into the chromosome of a host cell and towhich the polynucleotide is operably linked, into a host cell.

Then, the modification of the expression regulatory sequence forincreasing the expression of a polynucleotide (method 2) may beperformed by inducing a modification on the sequence through deletion,insertion, non-conservative or conservative substitution of thepolynucleotide sequence, or a combination thereof to further enhance theactivity of expression regulatory sequence, or by replacing thepolynucleotide sequence with a polynucleotide sequence having a strongeractivity, although the method is not particularly limited thereto. Theexpression regulatory sequence includes a promoter, an operatorsequence, a sequence coding for ribosome-binding site, and a sequenceregulating the termination of transcription and translation, althoughnot particularly limited thereto.

A strong exogenous promoter, instead of the original promoter, may beconnected to the upstream region of the expression unit of thepolynucleotide. Examples of the strong promoter may be CJ7 promoter,lysCP1 promoter, EF-Tu promoter, groEL promoter, aceA or aceB promoter,etc., and more specifically, the expression rate may be improved bybeing operably connected to Corynebacterium-derived lysCP1 promoter (WO2009/096689) or CJ7 promoter (Korean Patent No. 10-0620092 and WO2006/065095), but the strong promoter is not limited thereto.

Furthermore, the modification of a polynucleotide sequence on thechromosome (method 3) may be performed by inducing a modification on theexpression regulatory sequence through deletion, insertion,non-conservative or conservative substitution of the polynucleotidesequence, or a combination thereof to further enhance the activity ofthe polynucleotide sequence, or by replacing the polynucleotide sequencewith an improved polynucleotide sequence having a stronger activity,although the method is not particularly limited thereto.

Specifically, in the present disclosure, the activity of thephosphotransacetylase and acetate kinase operon (pta-ackA operon) may beenhanced in comparison with its endogenous activity by any one methodselected from the group consisting of a method of increasing the copynumber of the operon in a cell, a method of introducing a modificationon an expression regulatory sequence of the operon, a method ofreplacing an expression regulatory sequence of a gene on the operon witha sequence having a stronger activity, a method of replacing the genesencoding the enzymes with mutated genes on the chromosome for increasingthe activities of the enzymes constituting the operon, and a method ofintroducing a modification on the gene on the chromosome for increasingthe activities of the enzymes constituting the operon. Specifically, themethod of replacing an expression regulatory sequence of a gene on theoperon with a sequence having a stronger activity may be achieved byreplacing an endogenous promoter of the acetylase and acetate kinaseoperon with CJ7 promoter, lysCP1 promoter, EF-Tu promoter, groELpromoter, aceA or aceB promoter, etc., but the replacement is notlimited thereto.

As used herein, the term “endogenous activity” refers to an active stateof an enzyme in a non-modified state originally possessed by amicroorganism, and the term “enhancement compared to its endogenousactivity” refers to an increased state of the activity of the enzymepossessed by the microorganism after manipulation, such as theintroduction of a gene exhibiting an activity or an increase of the copynumber of the corresponding gene, deletion of the inhibition-regulatoryfactor of the expression of the gene, or modification of the expressionregulatory sequence, e.g., use of an improved promoter, compared to theactivity possessed by the microorganism before manipulation.

In the present disclosure, the microorganism of the genusCorynebacterium producing putrescine or ornithine, in which an activityof E. coli-derived acetyl-CoA synthetase (acs) may be further introducedtherein.

In the present disclosure, acetyl-CoA synthetase (acs) is an enzymewhich mediates the reaction for producing acetyl-CoA from ATP, aceticacid, and CoA.

In the present disclosure, the acetyl-CoA synthetase may include,without limitation, the proteins having the amino acid sequence of SEQID NO: 9, or any protein having a homology of 70% or higher,specifically 80% or higher, more specifically 90% or higher, even morespecifically 95% or higher, yet even more specifically 98% or higher,and most specifically 99% or higher, to the amino acid sequence above,as long as the protein has the substantial activity of mediating thesynthesis of acetyl-CoA.

Additionally, since the amino acid sequences of the proteins exhibitingthe activities may vary according to the species or strains of a givenmicroorganism, the acetyl-CoA synthetase (acs) in the present disclosuremay not be limited to the origin from which it is derived, and forexample, it may be from E. coli. It is obvious in that any amino acidsequence, which has a homology to the sequence above and has abiological activity substantially the same as or corresponding to theprotein of SEQ ID NO: 9, can also belong to the scope of the presentdisclosure, although the amino acid sequence may have deletion,modification, substitution, or addition, in part of the sequence.

The polynucleotide encoding the acetyl-CoA synthetase (acs) of thepresent disclosure may include the polynucleotide which encodes aprotein including the amino acid sequence of SEQ ID NO: 9, or anyprotein having a homology of 70% or higher, specifically 80% or higher,more specifically 90% or higher, even more specifically 95% or higher,yet even more specifically 98% or higher, and most specifically 99% orhigher, to the above amino acid sequence, and most specifically, it mayinclude the polynucleotide sequence of SEQ ID NO: 10.

The microorganism of the genus Corynebacterium of the present disclosuremay be a modified microorganism of the genus Corynebacterium producingputrescine or ornithine, in which an activity of ornithine decarboxylase(ODC) is further introduced therein.

As used herein, the term “ornithine decarboxylase” refers to an enzymewhich produces putrescine by mediating the decarboxylation of ornithine.Although the microorganism of the genus Corynebacterium lacks theputrescine biosynthetic enzyme, when ornithine decarboxylase (ODC) isintroduced from the outside, putrescine is exported outside the cell asputrescine is being synthesized. The ornithine decarboxylase that can beintroduced from the outside can be used in the present disclosure aslong as it has the activity above, irrespective of the origin from whichthe microorganism is derived, and specifically, one from E. coli may beintroduced.

The microorganism of the genus Corynebacterium of the present disclosuremay be a modified microorganism of the genus Corynebacterium producingputrescine or ornithine, in which, activities of i) ornithinecarbamoyltransferase (ArgF), ii) glutamate exporter, or iii) ornithinecarbamoyltransferase and glutamate exporter is further weakened,compared to its endogenous activity. The glutamate exporter of the genusCorynebacterium may be NCgl1221.

The microorganism of the genus Corynebacterium of the present disclosuremay be a modified microorganism of the genus Corynebacterium producingputrescine or ornithine, in which, an activity of at least one selectedfrom the group consisting of acetyl gamma glutamyl phosphate reductase(ArgC), acetylglutamate synthase or ornithine acetyltransferase (ArgJ),acetylglutamate kinase (ArgB), and acetyl ornithine aminotransferase(ArgD) is further enhanced compared to its endogenous activity.

Additionally, the microorganism of the genus Corynebacterium may be amodified microorganism of the genus Corynebacterium producing putrescineor ornithine, in which, an activity of acetyltransferase, specificallythe activity of NCgl1469, is further weakened in comparison with itsendogenous activity.

Lastly, the microorganism of the genus Corynebacterium may be a modifiedmicroorganism of the genus Corynebacterium producing putrescine orornithine, in which an activity of a putrescine exporter, specificallythe activity of NCgl2522, is further enhanced compared to its endogenousactivity.

As used herein, “weakening of activity” not only includes the drawing ofa lower effect than the original function due to the reduction orinactivation of the activity of a protein itself, but also includes thedecrease in its activity by a decrease in the activity of an endogenousgene, activation of regulatory factor(s) for inhibiting gene expression,a decrease in gene copy number, modification of the expressionregulatory sequence, and specifically, an inactivation or reduction inenzyme activity due to replacement or modification of a promoter and amutation within a gene, etc.

Specifically, in the present disclosure, the weakening of activity maybe performed by:

1) deleting a part or an entirety of a polynucleotide encoding theprotein,

2) modifying an expression regulatory sequence for reducing anexpression of the polynucleotide,

3) modifying a polynucleotide sequence on the chromosomes to weaken anactivity of the protein, and

4) a selected method from a combination thereof,

but the method is not limited thereto.

Specifically, the method of deleting a part or an entirety of apolynucleotide encoding a protein may be performed by replacing apolynucleotide encoding the endogenous target protein on the chromosomewith a polynucleotide having a partial deletion in the polynucleotidesequence or a marker gene using a vector for chromosomal insertionwithin bacteria. As used herein, the term “a part” may vary depending onthe kinds of polynucleotides, but it may specifically refer to 1 to 300,more specifically 1 to 100, and even more specifically 1 to 50.

Additionally, the method of modifying the expression regulatory sequencemay be performed by inducing a modification on the expression regulatorysequence through deletion, insertion, non-conservative or conservativesubstitution of a polynucleotide sequence, or a combination thereof tofurther weaken the activity of the expression regulatory sequence, or byreplacing the polynucleotide sequence with a polynucleotide sequencehaving a weaker activity. The expression regulatory sequence includes apromoter, an operator sequence, a sequence encoding a ribosome-bindingsite, and a sequence regulating the termination of transcription andtranslation.

Additionally, the method of modifying a polynucleotide sequence on thechromosome may be performed by inducing a modification on the sequencethrough deletion, insertion, non-conservative or conservativesubstitution of the polynucleotide sequence, or a combination thereof tofurther weaken the activity of the enzyme, or by replacing thepolynucleotide sequence with an improved polynucleotide sequence havinga stronger activity.

Additionally, the method of deleting the regulatory factor whichinhibits the expression of the polynucleotide of the enzyme may beperformed by replacing the polynucleotide for the expression inhibitingfactor with a polynucleotide having a partial deletion in thepolynucleotide sequence or a marker gene. As used herein, the term “apart” may vary depending on the kinds of polynucleotides, but it mayspecifically refer to 1 to 300, more specifically 1 to 100, and evenmore specifically 1 to 50.

In particular, acetyl gamma glutamyl phosphate reductase (ArgC),acetylglutamate synthase or ornithine acetyltransferase (ArgJ),acetylglutamate kinase (ArgB), acetylornithine aminotransferase (ArgD),ornithine carbamoyltransferase (ArgF), proteins involved in the exportof glutamate and ornithine decarboxylase (ODC) may respectively includethe amino acid sequence of SEQ ID NO: 32, 33, 34, 35, 36, 37, or 38, orany amino acid sequence, which specifically has a homology of 70% orhigher, more specifically 80% or higher, and even more specifically 90%or higher, to the above amino acid sequences, although not particularlylimited thereto. Additionally, the protein that acetylates putrescinemay include an amino acid sequence of SEQ ID NO: 30 or 31, or any aminoacid sequence, which specifically has a homology of 70% or higher, morespecifically 80% or higher, and even more specifically 90% or higher, tothe above amino acid sequences, although the amino acid sequence is notparticularly limited thereto.

Additionally, in the present disclosure, the putrescine exporter mayinclude an amino acid sequence of SEQ ID NO: 26 or 28, or any amino acidsequence, which specifically has a homology of 70% or higher, morespecifically 80% or higher, and even more specifically 90% or higher, tothe above amino acid sequences.

Among the proteins described above, the enhancement of activities ofacetyl gamma glutamyl phosphate reductase (ArgC), acetylglutamatesynthase or ornithineacetyltransferase (ArgJ), acetylglutamate kinase(ArgB), acetylornithine aminotransferase (ArgD), ornithine decarboxylase(ODC) and putrescine exporter may be achieved, for example, by a methodselected from an increase in copy number of the polynucleotides encodingthe proteins, modification of the expression regulatory sequence forincreasing the expression of the polynucleotides, modification of thepolynucleotide sequences on the chromosome for enhancing the activitiesof the above enzymes, deletion of regulatory factor(s) for inhibitingthe expression of the polynucleotides of the above enzymes, or acombination thereof.

Additionally, the weakening of ornithine carbamoyltransferase (ArgF),proteins involved in the export of glutamate, and the proteins thatacetylate putrescine may be achieved by a method selected from deletionof a part or the entirety of the polynucleotides encoding the proteins,modification of the expression regulatory sequence to reduce theexpression of the polynucleotides, modification of the polynucleotidesequences on the chromosome to weaken the activities of the proteins,and a combination thereof.

Another aspect of the present disclosure provides a method for producingputrescine or ornithine, including:

(i) culturing the microorganism of the genus Corynebacterium producingputrescine or ornithine in a medium; and

(ii) recovering putrescine or ornithine from the cultured microorganismor the medium.

In the above method, the microorganism may be cultured in batch culture,continuous culture, fed-batch culture, etc., known in the art, althoughnot particularly limited thereto. In particular, regarding the culturingcondition, proper pH (i.e., an optimal pH of 5 to 9, specifically pH 6to 8, and most specifically pH 6.8) can be maintained using a basiccompound (e.g., sodium hydroxide, potassium hydroxide, or ammonia) or anacidic compound (e.g., phosphoric acid or sulfuric acid), although notparticularly limited thereto. Additionally, an aerobic condition can bemaintained by adding oxygen or an oxygen-containing gas mixture to acell culture. The culture temperature may be maintained at 20° C. to 45°C., and specifically at 25° C. to 40° C., and the microorganism may becultured for about 10 hours to 160 hours. The putrescine or ornithineproduced by the culturing above may be secreted to a culture medium orremain in the cells.

Additionally, in the culture medium, carbon sources, such as sugars andcarbohydrates (e.g., glucose, sucrose, lactose, fructose, maltose,molasses, starch, and cellulose), oils and fats (e.g., soybean oil,sunflower seed oil, peanut oil, and coconut oil), fatty acids (e.g.,palmitic acid, stearic acid, and linoleic acid), alcohols (e.g.,glycerol and ethanol), and organic acids (e.g., acetic acid), may beused individually or in combination, but are not limited thereto;nitrogen sources, such as nitrogen-containing organic compounds (e.g.,peptone, yeast extract, meat juice, malt extract, corn steep liquor,soybean flour, and urea), or inorganic compounds (e.g., ammoniumsulfate, ammonium chloride, ammonium phosphate, ammonium carbonate, andammonium nitrate), may be used individually or in combination, but arenot limited thereto; and potassium sources, such as potassium dihydrogenphosphate, dipotassium hydrogen phosphate, or sodium-containing saltscorresponding thereto, may be used individually or in combination, butare not limited thereto. Additionally, other essentialgrowth-stimulating substances including metal salts (e.g., magnesiumsulfate or iron sulfate), amino acids, and vitamins may be furthercontained in the medium, but are not limited thereto.

The method of recovering the putrescine or ornithine produced during theculturing of the present disclosure may be performed by an appropriateculture method known in the art, for example, such as batch culture,continuous culture, or fed-batch culture, and thereby the target aminoacid can be recovered from the culture.

MODE FOR INVENTION

Hereinafter, the present disclosure will be described in more detailwith reference to the following Examples. However, these Examples arefor illustrative purposes only, and the disclosure is not intended to belimited by these Examples.

Example 1: Introduction of E. coli-Derived argA and E. coli-Derived argEinto a Strain Producing Putrescine and Confirmation ofPutrescine-Producing Ability of the Strain

1-1. Preparation of a Strain Simultaneously Introduced with E.coli-Derived argA and E. coli-Derived argE into a Transposon Gene ofATCC13032-Based Strain Producing Putrescine

In order to confirm whether the introduction of the E. coli-derived argAgene and the E. coli-derived argE gene into an ATCC13032-based strainproducing putrescine can improve putrescine-producing ability, argA andargE genes were introduced into the transposon gene of the strain.

As the vector for transformation enabling the introduction of thetransposon gene region of a microorganism of the genus Corynebacteriumwithin the chromosome, pDZTn (WO 2009/125992) was used, and lysCP1promoter (International Patent Publication No. WO 2009/096689, SEQ IDNO: 39) was used as the promoter.

Specifically, a primer pair of SEQ ID NOS: 11 and 12 for obtaining thehomologous recombinant fragments in the argA ORF region was preparedbased on the polynucleotide sequence (SEQ ID NO: 2) of the E.coli-derived argA gene, which encodes N-acetylglutamate synthase.Additionally, a primer pair of SEQ ID NOS: 15 and 16 for obtaining thehomologous recombinant fragments in the argE ORF region was preparedbased on the polynucleotide sequence (SEQ ID NO: 4) of the E.coli-derived argE gene, which encodes the acetylomithine deacetylase,and a primer pair of SEQ ID NOS: 13 and 14 for obtaining the homologousrecombinant fragments in the lysCP1 region was prepared based on thepolynucleotide sequence (SEQ ID NO: 39) of the lysCP1 (Table 1).

TABLE 1 Primer Sequence (5′→3′) PlysC-argA-FGAAAGGTGCACAAAGATGGTAAAGGAACGTAA (SEQ ID NO: 11) AACCG Tn-argA-RXhGCCCACTAGTCTCGAGCATGCGGCGTTGATTT (SEQ ID NO: 12) TG Tn-PlysC-FXhGAATGAGTTCCTCGAGCCGATGCTAGGGCGAA (SEQ ID NO: 13) AA PlysC-RCTTTGTGCACCTTTCGATCTACGTGCTGACAG (SEQ ID NO: 14) TTAC PlysC-argE-FGAAAGGTGCACAAAGATGAAAAACAAATTACC (SEQ ID NO: 15) GCC Tn-argE-RXhGCCCACTAGTCTCGAGGTTTGAGTCACTGTCG (SEQ ID NO: 16) GTCG

First, a gene fragment with a size of about 1.6 kb was amplified usingthe chromosome of E. coli W3110 strain as the template along with aprimer pair of SEQ ID NOS: 11 and 12, in order to obtain the argA gene.In particular, PCR was performed by repeating 30 cycles of denaturationat 95° C. for 30 seconds, annealing at 55° C. for 30 seconds, andextension at 72° C. for 1 minute and 30 seconds. The thus-obtainedfragments were subjected to electrophoresis in a 0.8% agarose gel, andthe bands of desired sizes were eluted and purified.

Additionally, the lysCP1 promoter region was by performing PCR using thechromosome of the KCCM10919P (International Patent Publication No. WO2009/096689) strain as the template along with a primer pair of SEQ IDNOS: 13 and 14, which was performed by repeating 30 cycles ofdenaturation at 95° C. for 30 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 30 seconds.

The pDZTn vector was treated with XhoI and then each of the PCR productsobtained thereof was subjected to fusion cloning. The fusion cloning wasperformed using the In-Fusion® HD Cloning Kit (Clontech) and thethus-obtained plasmid was named as pDZTn-lysCP1-argA.

Then, for obtaining the argE gene, PCR products were obtained byamplifying the gene fragment with a size of about 1.4 kb in the samemanner as described above, using the chromosome of the E. coli W3110strain as the template along with a primer pair of SEQ ID NOS: 15 and16, and was subjected to fusion cloning with the lysCP1 promoter region.The thus-obtained plasmid was named as pDZTn-lysCP1-argE.

Then, the plasmid pDZTn-lysCP1-argA was introduced into the KCCM11240P(Korean Patent Application Publication No. 10-2013-0082478) strain byelectroporation to obtain transformants, and the transformants wereplated on BHIS plate media (Braine heart infusion (37 g/L), sorbitol (91g/L), and agar (2%)) containing kanamycin (25 μg/mL) and X-gal(5-bromo-4-chloro-3-indolin-D-galactoside) and cultured to formcolonies. Among the colonies, blue colonies were selected and therebythe transformed strains introduced with the plasmid pDZTn-lysCP1-argAwere selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strain introduced with the argA-encoding gene by asecondary crossover was finally selected. The finally selected strainwas subjected to PCR using a primer pair of SEQ ID NOS: 12 and 13 and itwas confirmed that the argA-encoding gene was introduced, and themodified strain of Corynebacterium glutamicum was named as KCCM11240PTn:lysCP1-argA.

For the introduction of the strain introduced with argA prepared above,the pDZTn-lysCP1-argE prepared above was transformed into the KCCM11240PTn:lysCP1-argA in the same manner as described above, and theintroduction of argE into the transposon was confirmed in the finallyselected strain by performing PCR using a primer pair of SEQ ID NOS: 13and 16. The thus-selected modified strain of Corynebacterium glutamicumwas named as KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE.

1-2. Preparation of a Strain Simultaneously Introduced with E.coli-Derived argA and E. coli-Derived argE into a Transposon Gene ofATCC13869-Based Strain Producing Putrescine

The DAB12-a ΔNCgl1469 (Korean Patent Application Publication No.10-2013-0082478), which is a Corynebacterium glutamicum ATCC13869-basedstrain producing putrescine, was named as DAB12-b, and argA and argEwere introduced into the transposon gene in order to confirm whether theintroduction of the E. coli-derived argA and E. coli-derived argE genescan be associated with the improvement of the putrescine-producingability of the resulting strain.

First, the pDZTn-lysCP1-argA, which was previously prepared, wastransformed into the Corynebacterium glutamicum DAB12-b in the samemanner as in Example 1-1, and the introduction of argA into thetransposon was confirmed. The thus-selected modified strain ofCorynebacterium glutamicum was named as DAB12-b Tn:lysCP1-argA.

Then, for the introduction of argE into the strain, which is alreadyintroduced with argA, the pDZTn-lysCP1-argE, which was previouslyprepared, was transformed into the DAB12-b Tn:lysCP1-argA in the samemanner as in Example 1-1, and the introduction of argE into thetransposon was confirmed. The thus-selected modified strain ofCorynebacterium glutamicum was named as DAB12-b Tn:lysCP1-argE.

1-3. Evaluation of Putrescine-Producing Ability of a CorynebacteriumStrain Producing Putrescine Introduced with E. coli-Derived argA Geneand E. coli-Derived argE Gene

The putrescine-producing ability was compared among the modified strainsof Corynebacterium glutamicum prepared in Examples 1-1 and 1-2, in orderto confirm the effect of the introduction of the E. coli-derived argAand the E. coli-derived argE into a strain producing putrescine onputrescin production.

Specifically, two different kinds of modified strains of Corynebacteriumglutamicum, i.e., (KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE; DAB12-bTn:lysCP1-argA Tn:lysCP1-argE) prepared in Examples 1-1 and 1-2, and twodifferent kinds of parent strains (i.e., KCCM11240P and DAB12-b) wererespectively plated on 1 mM arginine-containing CM plate media (1%glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25%NaCl, 0.2% urea, 100 μL of 50% NaOH, 2% agar, pH 6.8, based on 1 L), andcultured at 30° C. for 24 hours.

Each of the strains cultured therefrom in an amount of about oneplatinum loop was inoculated into 25 mL of titer media (8% glucose,0.25% soybean protein, 0.50% corn steep solids, 4% (NH₄)₂SO₄, 0.1%KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100 μg), thiamine HCl (3mg), calcium-pantothenic acid (3 mg), nicotinamide (3 mg), 5% CaCO₃,based on 1 L), and cultured with shaking at 30° C. at a rate of 200 rpmfor 98 hours. In all cultures of the strains, 1 mM arginine was added tothe media. Upon completion of culture, the concentration of putrescineproduced in each culture broth was measured and the results are shown inTable 2 below.

TABLE 2 Strains Putrescine (g/L) KCCM 11240P 12.2 KCCM11240PTn:lysCP1-argA Tn:lysCP1-argE 13.4 DAB12-b 13.3 DAB12-b Tn:lysCP1-argATn:lysCP1-argE 14.6

As shown in Table 2 above, both of the two modified strains ofCorynebacterium glutamicum simultaneously introduced with E.coli-derived argA and E. coli-derived argE genes showed an increase ofputrescine production by 9.8% or higher.

Example 2: Enhancement of Pta-ackA in the Strain Producing PutrescineIntroduced with E. coli-Derived argA and E. coli-Derived argE andConfirmation of Putrescine-Producing Ability of the Strain

2-1. Preparation of a Strain Having a Substitution of the Pta-ackAPromoter from an ATCC13032-Based Corynebacterium Strain ProducingPutrescine

The strain producing putrescine introduced with E. coli-derived argA andE. coli-derived argE genes, prepared in Example 1, was further enhancedin its activity of phosphotransacetylase and acetate kinase (pta-ackA)and the effect of the enhancement on the putrescine-producing ability ofthe strain was examined.

For this purpose, the promoter of the pta-ackA operon within thechromosome was substituted with a promoter having a stronger activity incomparison with its endogenous promoter, specifically, the lysCP1promoter (International Patent Publication No. WO 2009/096689) wasintroduced to the upstream of the initiation codon of the pta-ackAoperon.

First, a homologous recombinant fragment, which includes the lysCP1promoter and both ends of the promoter have the original pta-ackAsequence on the chromosome, was obtained. Specifically, the 5′-endregion of the lysCP1 promoter was obtained by performing PCR using thegenomic DNA of the Corynebacterium glutamicum ATCC13032 along with aprimer pair of SEQ ID NOS: 17 and 18. In particular, PCR reaction wasperformed by repeating 30 cycles of denaturation at 95° C. for 30seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for30 seconds.

Additionally, the lysCP1 promoter region was obtained by performing PCRin the same condition using a primer pair of SEQ ID NOS: 14 and 19, andthe 3′-end region of the lysCP1 promoter was obtained by performing PCRusing the genomic DNA of the Corynebacterium glutamicum ATCC13032 as atemplate along with a primer pair of SEQ ID NOS: 20 and 21. The primersused in obtaining the lysCP1 promoter are shown in Table 1 above andTable 3 below.

TABLE 3 Primer Sequence (5′→3′) Pro-pta-FXCCGGGGATCCTCTAGAGGGGTTCTAAAAAATG (SEQ ID NO: 17) TGGAGT pta-Ply sC-RGCCGTGCTTTTCGCCCTAGCATCGGACATCGC (SEQ ID NO: 18) CTTTCTAATTT PlysC-FCCGATGCTAGGGCGAAAAGCACGGC (SEQ ID NO: 19) PlysC-pta-ackA-FGAAAGGTGCACAAAGATGTCTGACACACCGAC (SEQ ID NO: 20) CTCAGCTC Pro-pta-RXGCAGGTCGACTCTAGATTATCCGGCATTGGCT (SEQ ID NO: 21) CT

Each of the PCR products obtained above was subjected to fusion cloningusing the pDZ vector treated with XbaI. The fusion cloning was performedusing the In-Fusion® HD Cloning Kit (Clontech) and the thus-obtainedplasmid was named as pDZ-lysCP1-1′pta-ackA.

The plasmid pDZ-lysCP1-1′pta-ackA prepared from the above wasrespectively introduced into the KCCM11240P and KCCM11240PTn:lysCP1-argA Tn:lysCP1-argE strains, which is a modified strain ofCorynebacterium glutamicum prepared in Example 1-1, by electroporationto obtain transformants, and the transformants were plated on BHIS platemedia (Braine heart infusion (37 g/L), sorbitol (91 g/L), and agar (2%))containing kanamycin (25 μg/mL) and X-gal(5-bromo-4-chloro-3-indolin-D-galactoside) and cultured to formcolonies. Among the colonies, blue colonies were selected and therebythe transformed strains introduced with the plasmidpDZ-lysCP1-1′pta-ackA were selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strain, in which the pta-ackA promoter was substitutedwith the lysCP1 promoter by a secondary crossover, was finally selected.

The finally selected strain was subjected to PCR using a primer pair ofSEQ ID NOS: 19 and 21 and was confirmed that the lysCP1 promoter wasintroduced to the upstream of the initiation codon of pta-ackA withinthe chromosome. In particular, the PCR reaction was performed byrepeating 30 cycles of denaturation at 95° C. for 30 seconds, annealingat 55° C. for 30 seconds, and extension at 72° C. for 1 minute.

The thus-selected modified strains of Corynebacterium glutamicum werenamed as KCCM11240P lysCP1-1′pta-ackA and KCCM11240P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA, respectively.

2-2. Preparation of a Strain Having a Substitution of the Pta-ackAPromoter from an ATCC13869-Based Corynebacterium Strain ProducingPutrescine

In order to confirm the sequence of the gene encoding the pta-ackAderived from Corynebacterium glutamicum ATCC13869 and the proteinexpressed therefrom by the method disclosed in Example 2-1, PCR wasperformed using the genomic DNA of Corynebacterium glutamicum ATCC13869as a template along with a primer pair of SEQ ID NOS: 17 and 22 (Tables3 and 4). In particular, the PCR reaction was performed by repeating 30cycles of denaturation at 95° C. for 30 seconds, annealing at 55° C. for30 seconds, and extension at 72° C. for 3 minutes.

The thus-obtained PCR products were separated by electrophoresis and thesequences were analyzed. As a result, it was confirmed that the geneencoding the pta-ackA derived from Corynebacterium glutamicum ATCC13869includes a polynucleotide sequence described by SEQ ID NO: 8 and thatthe protein encoded by the gene includes an amino acid sequencedescribed by SEQ ID NO: 7.

On the other hand, as a result of the comparison between the amino acidsequence of pta-ackA derived from Corynebacterium glutamicum ATCC13032(SEQ ID NO: 5) and the amino acid sequence of pta-ackA derived fromCorynebacterium glutamicum ATCC13869, it was confirmed that they have asequence homology of 99.4%.

TABLE 4 Primer Sequence (5′→3′) Pta-ackA-R TGCAGTTTCACCCCTTAA(SEQ ID NO: 22) 13869_pta-PlysC-R GCCGTGCTTTTCGCCCTAGCATCGGACATCG(SEQ ID NO: 23) CCTTTCTAGTTT

First, a homologous recombinant fragment, which includes the lysCP1promoter and both ends of the promoter have the original pta-ackAsequence on the chromosome, was obtained. Specifically, the 5′-endregion of the lysCP1 promoter was obtained by performing PCR using thegenomic DNA of the Corynebacterium glutamicum ATCC13869 along with aprimer pair of SEQ ID NOS: 17 and 23. In particular, PCR reaction wasperformed by repeating 30 cycles of denaturation at 95° C. for 30seconds, annealing at 55° C. for 30 seconds, and extension at 72° C. for30 seconds. Additionally, the lysCP1 promoter region was obtained byperforming PCR in the same condition using a primer pair of SEQ ID NOS:14 and 19, and the 3′-end region of the lysCP1 promoter was obtained byperforming PCR using the genomic DNA of the Corynebacterium glutamicumATCC13869 as a template along with a primer pair of SEQ ID NOS: 20 and21. The primers used in the promoter substitution are shown in Tables 1,3 and 4.

Each of the PCR products obtained thereof was subjected to fusioncloning using the pDZTn vector treated with XhoI. The fusion cloning wasperformed using the In-Fusion® HD Cloning Kit (Clontech) and thethus-obtained plasmid was named as pDZ-lysCP1-2′pta-ackA.

The plasmid pDZ-lysCP1-2′pta-ackA prepared from the above wasrespectively transformed into DAB12-b and DAB12-b Tn:lysCP1-argATn:lysCP1-argE, which is a modified strain of Corynebacterium glutamicumprepared in Example 1-2, in the same manner as in Example 2-1. As aresult, it was confirmed that the lysCP1 promoter was introduced to theupstream of the initiation codon of pta-ackA within the chromosome. Themodified strains of Corynebacterium glutamicum were named as DAB12-blysCP1-2′pta-ackA and DAB12-b Tn:lysCP1-argA Tn:lysCP1-argElysCP1-2′pta-ackA, respectively.

2-3. Evaluation of Putrescine-Producing Ability of a Strain withEnhanced Pta-ackA

In order to confirm the effect of the enhancement of pta-ackA in astrain producing putrescine introduced with E. coli-derived argA and E.coli-derived argE, the putrescine-producing ability was compared amongthe modified strains of Corynebacterium glutamicum prepared in Examples2-1 and 2-2.

Specifically, four kinds of modified strains of Corynebacteriumglutamicum (KCCM11240P lysCP1-1′pta-ackA; KCCM11240P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA; DAB12-b lysCP1-2′pta-ackA; and DAB12-bTn:lysCP1-argA Tn:lysCP1-argE lysCP1-2′pta-ackA) and four kinds ofparent strains (KCCM11240P; KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE;DAB12-b; and DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE) were respectivelyplated on 1 mM arginine-containing CM plate media (1% glucose, 1%polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2%urea, 100 μL of 50% NaOH, 2% agar, pH 6.8, based on 1 L), and culturedat 30° C. for 24 hours. Each of the strains cultured therefrom in anamount of about one platinum loop was inoculated into 25 mL of titermedia (8% glucose, 0.25% soybean protein, 0.50% corn steep solids, 4%(NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100 μg),thiamine HCl (3 mg), calcium-pantothenic acid (3 mg), nicotinamide (3mg), 5% CaCO₃, based on 1 L), and cultured with shaking at 30° C. at arate of 200 rpm for 98 hours. In all cultures of the strains, 1 mMarginine was added to the media. Upon completion of culture, theconcentration of putrescine produced in each culture broth was measuredand the results are shown in Table 5 below.

TABLE 5 Strains Putrescine (g/L) KCCM 11240P 12.2 KCCM 11240PlysCP1-1′pta-ackA 12.3 KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE 13.4KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE 14.1 lysCP1-1′pta-ackA DAB12-b13.3 DAB12-b lysCP1-2′pta-ackA 13.4 DAB12-b Tn:lysCP1-argATn:lysCP1-argE 14.6 DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE 15.2lysCP1-2′pta-ackA

As shown in Table 5, when pta-ackA was enhanced in KCCM 11240P andDAB12-b, respectively, the amount of putrescine production was at thesame level. However, when pta-ackA was enhanced in the two differentkinds of modified strains of Corynebacterium glutamicum simultaneouslyintroduced with E. coli-derived argA and E. coli-derived argE genes(KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE; DAB12-b Tn:lysCP1-argATn:lysCP1-argE), respectively, the amount of putrescine production wasincreased by 14.3% or higher, compared to the parent strain.Additionally, the amount of putrescine production was increased by 4% orhigher, based on the modified strains.

As such, the present inventors named the microorganism of the genusCorynebacterium (Corynebacterium glutamicum KCCM11240P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA), which has an improved ability toproduce putrescine, prepared from the Corynebacterium glutamicum KCCM11240P strain producing putrescine by introducing the activities of E.coli-derived argA and E. coli-derived argE and enhancing the activity ofpta-ackA to the Corynebacterium glutamicum KCCM 11240P strain, asCC01-1145, and deposited in the Korean Culture Center of Microorganisms(KCCM), (Address: Yurim B/D, 45, Hongjenae-2ga-gil, Seodaemun-gu, SEOUL120-861, Republic of Korea), on Nov. 21, 2014, with the accession numberKCCM11606P under the Budapest Treaty on the International Recognition ofthe Deposit of Microorganisms for the Purposes of Patent Procedure.

Example 3: Introduction of E. coli-Derived Acs into a Strain ProducingPutrescine Introduced with E. coli-Derived argA and E. coli-Derived argEand Confirmation of the Putrescine-Producing Ability of the ResultingStrain

3-1. Preparation of a Strain Introduced with E. coli-Derived Acs into aTransposon Gene of an ATCC13032-Based Strain Producing Putrescine

The acs was introduced into the transposon gene using the lysCP1promoter in order to confirm whether the introduction of E. coli-derivedacetyl-CoA synthetase (acs) gene into an ATCC13032-based strainproducing putrescine, which is already introduced with E. coli-derivedargA and E. coli-derived argE, can improve the putrescine-producingability.

Specifically, a primer pair of SEQ ID NOS: 24 and 25 for obtaining thehomologous recombinant fragment around the acs ORF region and a primerpair of SEQ ID NOS: 13 and 14 for obtaining the homologous recombinantfragment around the lysCP1 promoter region were prepared as shown inTable 1 above and Table 6 below, based on the polynucleotide sequencedescribed by SEQ ID NO: 10 of the gene encoding the acs.

TABLE 6 Primer Sequence (5′→3′) PlysC-acs-FGAAAGGTGCACAAAGATGAGCCAAATTCACAAA (SEQ ID NO: 24) Tn-acs-RXhGCCCACTAGTCTCGAGAAGGCGTTTACGCCGCA (SEQ ID NO: 25) TCC

Specifically, for obtaining the acs gene, the gene fragment with a sizeof about 2 kb was amplified using the chromosome of the E. coli W3110strain as a template along with a primer pair of SEQ ID NOS: 24 and 25.In particular, PCR reaction was performed by repeating 30 cycles ofdenaturation at 95° C. for 30 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 1 minute and 30 seconds. Then, thethus-obtained PCR products were subjected to electrophoresis in a 0.8%agarose gel and the bands of desired sizes were eluted and purified.

Additionally, the lysCP1 promoter region was obtained by performing PCRusing the chromosome of the KCCM10919P (International Patent PublicationNo. WO 2009/096689) strain as the template along with a primer pair ofSEQ ID NOS: 13 and 14, which was performed by repeating 30 cycles ofdenaturation at 95° C. for 30 seconds, annealing at 55° C. for 30seconds, and extension at 72° C. for 30 seconds.

The pDZ vector was treated with XhoI and each of the thus-obtained PCRproducts was subjected to fusion cloning. The fusion cloning wasperformed using the In-Fusion® HD Cloning Kit (Clontech). Thethus-obtained plasmid was named as pDZTn-lysCP1-acs.

Then, the plasmid pDZTn-lysCP1-acs was introduced into the KCCM11240Pand KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE, which is a modified strainof Corynebacterium glutamicum prepared in Example 1-1, respectively, byelectroporation to obtain transformants, and the transformants wereplated on BHIS plate media (Braine heart infusion (37 g/L), sorbitol (91g/L), and agar (2%)) containing kanamycin (25 μg/mL) and X-gal(5-bromo-4-chloro-3-indolin-D-galactoside) and cultured to formcolonies. Among the colonies, blue colonies were selected and therebythe transformed strains introduced with the plasmid pDZTn-lysCP1-acswere selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strains introduced with the acs-encoding gene by asecondary crossover were finally selected. The finally selected strainswere subjected to PCR using a primer pair of SEQ ID NOS: 13 and 25 andconfirmed that the acs-encoding gene was introduced, and the modifiedstrains of Corynebacterium glutamicum were named as KCCM11240PTn:lysCP1-acs and KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argETn:lysCP1-acs, respectively.

3-2. Preparation of a Strain Introduced with E. coli-Derived Acs into aTransposon Gene of ATCC13869-Based Strain Producing Putrescine

As in Example 3-1, the pDZTn-lysCP1-acs prepared from the above wastransformed into DAB12-b and the DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE,which is a modified strain of Corynebacterium glutamicum prepared inExample 1-2, respectively, in the same manner as in Example 3-1, and itwas confirmed that the acs was introduced into the transposon gene.

The thus-selected modified strains of Corynebacterium glutamicum werenamed as DAB12-b Tn:lysCP1-acs and DAB12-b Tn:lysCP1-argA Tn:lysCP1-argETn:lysCP1-acs, respectively.

3-3. Evaluation of Putrescine-Producing Ability of a Strain Introducedwith E. coli-Derived Acs

In order to confirm the effect of the introduction of acs in a strainproducing putrescine, which is already introduced with E. coli-derivedargA and E. coli-derived argE, putrescine-producing ability was comparedamong the modified strains of Corynebacterium glutamicum prepared inExamples 3-1 and 3-2.

Specifically, four kinds of modified strains of Corynebacteriumglutamicum (KCCM11240P Tn:lysCP1-acs; KCCM11240P Tn:lysCP1-argATn:lysCP1-argE Tn:lysCP1-acs; DAB12-b Tn:lysCP1-acs; and DAB12-bTn:lysCP1-argA Tn:lysCP1-argE Tn:lysCP1-acs) and four kinds of parentstrains (KCCM11240P; KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE; DAB12-b;and DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE) were respectively plated on 1mM arginine-containing CM plate media (1% glucose, 1% polypeptone, 0.5%yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μL of 50%NaOH, 2% agar, pH 6.8, based on 1 L), and cultured at 30° C. for 24hours. Each of the strains cultured therefrom in an amount of about oneplatinum loop was inoculated into 25 mL of titer media (8% glucose,0.25% soybean protein, 0.50% corn steep solids, 4% (NH₄)₂SO₄, 0.1%KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100 μg), thiamine HCl (3mg), calcium-pantothenic acid (3 mg), nicotinamide (3 mg), 5% CaCO₃,based on 1 L), and cultured with shaking at 30° C. at a rate of 200 rpmfor 98 hours. In all cultures of the strains, 1 mM arginine was added tothe media. Upon completion of culture, the concentration of putrescineproduced in each culture broth was measured and the results are shown inTable 7 below.

TABLE 7 Strains Putrescine (g/L) KCCM 11240P 12.2 KCCM 11240PTn:lysCP1-acs 12.2 KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE 13.4KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE 13.9 Tn:lysCP1-acs DAB12-b 13.3DAB12-b Tn:lysCP1-acs 13.2 DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE 14.6DAB12-b Tn:lysCP1-argA Tn:lysCP1-argE 15.1 Tn:lysCP1-acs

As shown in Table 7, when acs was introduced into KCCM 11240P andDAB12-b, respectively, the amount of putrescine production was at thesame level. However, when acs was introduce in the two different kindsof modified strains of Corynebacterium glutamicum simultaneouslyintroduced with E. coli-derived argA and E. coli-derived argE genes(KCCM11240P Tn:lysCP1-argA Tn:lysCP1-argE; DAB12-b Tn:lysCP1-argATn:lysCP1-argE), respectively, the amount of putrescine production wasincreased by 13.5% or higher, compared to the parent strain.Additionally, the amount of putrescine production was increased by 3.4%or higher, compared to the above modified strains.

Example 4: A Strain Having Introduction of E. coli-Derived argA, E.coli-Derived argE, and Substitution of Pta-ackA Promoter from a StrainProducing Putrescine with Improved Putrescine Export Ability, and thePutrescine-Producing Ability of the Strain

4-1. Preparation of a Strain Having Introduction of E. coli-DerivedargA, -argE and Substitution of Pta-ackA Promoter from a Strain HavingImproved Putrescine Export Ability

A strain was prepared to examine whether the introduction of E.coli-derived argA and E. coli-derived argE and the enhancement of theactivity of the Corynebacterium pta-ackA can improve theputrescine-producing ability, based on the KCCM11401P (Korean PatentApplication Publication No. 10-2014-0115244) strain with improvedputrescine export ability.

Specifically, the pDZTn-lysCP1-argA prepared in Example 1-1 wastransformed into the KCCM11401P in the same manner as in Example 1-1,and as a result, it was confirmed that argA was introduced into thetransposon gene. The thus-selected modified strain of Corynebacteriumglutamicum was named as KCCM11401P Tn:lysCP1-argA.

Additionally, for introducing argE into the strain, which is alreadyintroduced with argA as prepared in Example 1-1, the pDZTn-lysCP1-argEprepared in Example 1-1 was transformed into the KCCM11401PTn:lysCP1-argA in the same manner as in Example 1-1 and it was confirmedthat argE was introduced into the transposon gene. The thus-selectedmodified strain was named as KCCM11401P Tn:lysCP1-argA Tn:lysCP1-argE.

Then, the pDZ-lysCP1-1′pta-ackA prepared in Example 2-1 was transformedinto the KCCM11401P Tn:lysCP1-argA Tn:lysCP1-argE in the same manner asin Example 2-1, and it was confirmed that the lysCP1 promoter wasintroduced to the upstream of the initiation codon of pta-ackA withinthe chromosome. The above modified strain of Corynebacterium glutamicumwas named as KCCM11401P Tn:lysCP1-argA Tn:lysCP1-argE lysCP1-1′pta-ackA.

4-2. Evaluation of a Strain Having Introduction of E. coli-Derived argA,E. coli-Derived argE and Substitution of Pta-ackA Promoter from a StrainHaving Improved Putrescine Export Ability

In order to confirm the effect of the introduction of E. coli-derivedargA and E. coli-derived argE and the enhancement of pta-ackA activityon a strain of Corynebacterium glutamicum producing putrescine withimproved putrescine export ability, the putrescine-producing ability wascompared among the modified strains of Corynebacterium glutamicumprepared in Example 4-1.

Specifically, the modified strains of Corynebacterium glutamicum(KCCM11401P Tn:lysCP1-argA Tn:lysCP1-argE, KCCM11401P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA) and the parent strain (KCCM11401P)were respectively plated on 1 mM arginine-containing CM plate media (1%glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25%NaCl, 0.2% urea, 100 μL of 50% NaOH, 2% agar, pH 6.8, based on 1 L), andcultured at 30° C. for 24 hours. Each of the strains cultured therefromin an amount of about one platinum loop was inoculated into 25 mL oftiter media (8% glucose, 0.25% soybean protein, 0.50% corn steep solids,4% (NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100μg), thiamine HCl (3 mg), calcium-pantothenic acid (3 mg), nicotinamide(3 mg), 5% CaCO₃, based on 1 L), and cultured with shaking at 30° C. ata rate of 200 rpm for 98 hours. In all cultures of the strains, 1 mMarginine was added to the media. Upon completion of culture, theconcentration of putrescine produced in each culture broth was measuredand the results are shown in Table 8 below.

TABLE 8 Strains Putrescine (g/L) KCCM11401P 11.8 KCCM11401PTn:lysCP1-argA Tn:lysCP1-argE 13.2 KCCM11401P Tn:lysCP1-argATn:lysCP1-argE 13.7 lysCP1-1′pta-ackA

As shown in Table 8, it was confirmed that when the KCCM11401P havingenhanced putrescine export ability was introduced with E. coli-derivedargA gene and E. coli-derived argE gene, the amount of putrescineproduction was increased by 11.9% compared to that of the partentstrain, and when the strain was further enhanced with pta-ackA, theamount of putrescine production was increased by 16.1% compared to thatof the partent strain.

Example 5: Introduction of E. coli-Derived argA and E. coli-Derived argEin a Strain Producing Ornithine and Confirmation of theOrnithine-Producing Ability of the Strain

5-1. Preparation of a Strain Simultaneously Introduced with E.coli-Derived argA and E. coli-Derived argE into a Transposon Gene ofKCCM11137P-Based Strain Producing Omithine

In order to confirm whether the introduction of E. coli-derived argAgene and E. coli-derived argE gene into the KCCM11137P (Korean PatentApplication Publication No. 10-1372635) strain, which is aCorynebacterium glutamicum ATCC13032-based strain producing omithine,can improve ornithine-producing ability, argA gene and argE gene wereintroduced into a transposon gene of the strain using the vectorprepared in Example 1-1.

First, the plasmid pDZTn-lysCP1-argA was introduced into the KCCM11137Pstrain by electroporation to obtain transformants, and the transformantswere plated on BHIS plate media (Braine heart infusion (37 g/L),sorbitol (91 g/L), and agar (2%)) containing kanamycin (25 μg/mL) andX-gal (5-bromo-4-chloro-3-indolin-D-galactoside) and cultured to formcolonies. Among the colonies, blue colonies were selected and therebythe strains introduced with the plasmid pDZTn-lysCP1-argA were selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strain introduced with the argA-encoding gene by asecondary crossover was finally selected. The finally selected strainwas subjected to PCR using a primer pair of SEQ ID NOS: 12 and 13 andconfirmed that the argA-encoding gene was introduced, and the modifiedstrain of Corynebacterium glutamicum was named as KCCM11137PTn:lysCP1-argA.

For the introduction of argE into the strain, which is alreadyintroduced with argA as prepared above, the pDZTn-lysCP1-argE preparedin Example 1-1 was transformed into the KCCM11137P Tn:lysCP1-argA in thesame manner as in Example 1-1, and thereby it was confirmed that theargE was introduced within the transposon gene.

The thus-selected modified strain of Corynebacterium glutamicum wasnamed as KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE.

5-2. Evaluation of Ornithine-Producing Ability of a CorynebacteriumStrain Producing Ornithine Introduced with E. coli-Derived argA and E.coli-Derived argE

In order to confirm the effect of the introduction of E. coli-derivedargA and E. coli-derived argE on ornithine production in a strainproducing ornithine, the ornithine-producing ability was compared amongthe modified strains of Corynebacterium glutamicum prepared in Example5-1.

Specifically, one kind of a modified strain of Corynebacteriumglutamicum (KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE) and one kind of aparent strain (KCCM11137P) were respectively plated on 1 mMarginine-containing CM plate media (1% glucose, 1% polypeptone, 0.5%yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2% urea, 100 μL of 50%NaOH, 2% agar, pH 6.8, based on 1 L), and cultured at 30° C. for 24hours. Each of the strains cultured therefrom in an amount of about oneplatinum loop was inoculated into 25 mL of titer media (8% glucose,0.25% soybean protein, 0.50% corn steep solids, 4% (NH₄)₂SO₄, 0.1%KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100 μg), thiamine HCl (3mg), calcium-pantothenic acid (3 mg), nicotinamide (3 mg), 5% CaCO₃,based on 1 L), and cultured with shaking at 30° C. at a rate of 200 rpmfor 98 hours. In all cultures of the strains, 1 mM arginine was added tothe media. Upon completion of culture, the concentration of putrescineproduced in each culture broth was measured and the results are shown inTable 9 below.

TABLE 9 Strains Ornithine (g/L) KCCM11137P 7.8 KCCM11137P Tn:lysCP1-argATn:lysCP1-argE 8.9

As shown in Table 9, it was confirmed that when the modified strain ofCorynebacterium glutamicum introduced with E. coli-derived argA gene andE. coli-derived argE gene showed an increase in the amount of ornithineproduction by 14.1% compared to that of the partent strain.

Example 6: Enhancement of Pta-ackA in a Strain Introduced with E.coli-Derived argA and E. coli-Derived argE and Confirmation ofOrnithine-Producing Ability of the Strain

6-1. Preparation of a Strain Having a Substitution of Pta-ackA Promoterfrom an ATCC13032-Based Strain Producing Ornithine

In order to confirm whether the enhancement of pta-ackA activity intothe ATCC13032-based strain producing ornithine introduced with E.coli-derived argA and E. coli-derived argE can improve theornithine-producing ability, the lysCP1 promoter (WO 2009/096689) wasintroduced to the upstream of the initiation codon of pta-ackA operonwithin the chromosome.

First, the plasmid pDZ-lysCP1-1′pta-ackA prepared in Example 2-1 wasintroduced into KCCM11137P and KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argEstrains, respectively, by electroporation to obtain transformants andthe transformants were plated on BHIS plate media (Braine heart infusion(37 g/L), sorbitol (91 g/L), and agar (2%)) containing kanamycin (25μg/mL) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) and culturedto form colonies. Among the colonies, blue colonies were selected andthereby the transformed strains introduced with the plasmidpDZ-lysCP1-1′pta-ackA were selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strain, in which the pta-ackA promoter was substitutedwith the lysCP1 promoter by a secondary crossover, was finally selected.The finally selected strain was subjected to PCR using a primer pair ofSEQ ID NOS: 19 and 21 and confirmed that the lysCP1 promoter wasintroduced to the upstream of the initiation codon of pta-ackA operonwithin the chromosome. In particular, PCR reaction was performed byrepeating 30 cycles of denaturation at 95° C. for 30 seconds, annealingat 55° C. for 30 seconds, and extension at 72° C. for 1 minute.

The thus-selected modified strains of Corynebacterium glutamicum werenamed as KCCM11137P lysCP1-1′pta-ackA and KCCM11137P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA, respectively.

6-2. Evaluation of Ornithine-Producing Ability of a Strain with EnhancedPta-ackA Activity

In order to confirm the effect of the enhancement of pta-ackA activityon a strain producing ornithine introduced with E. coli-derived argA andE. coli-derived argE, the ornithine-producing ability was compared amongthe modified strains of Corynebacterium glutamicum prepared in Example6-1.

Specifically, two different kinds of modified strains of Corynebacteriumglutamicum, (KCCM11137P lysCP1-1′pta-ackA; KCCM11137P Tn:lysCP1-argATn:lysCP1-argE lysCP1-1′pta-ackA) and two different kinds of parentstrains (KCCM11137P; KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE) wererespectively plated on 1 mM arginine-containing CM plate media (1%glucose, 1% polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25%NaCl, 0.2% urea, 100 μL of 50% NaOH, 2% agar, pH 6.8, based on 1 L), andcultured at 30° C. for 24 hours. Each of the strains cultured therefromin an amount of about one platinum loop was inoculated into 25 mL oftiter media (8% glucose, 0.25% soybean protein, 0.50% corn steep solids,4% (NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100μg), thiamine HCl (3 mg), calcium-pantothenic acid (3 mg), nicotinamide(3 mg), 5% CaCO₃, based on 1 L), and cultured with shaking at 30° C. ata rate of 200 rpm for 98 hours. In all cultures of the strains, 1 mMarginine was added to the media. Upon completion of culture, theconcentration of ornithine produced in each culture broth was measuredand the results are shown in Table 10 below.

TABLE 10 Strains Ornithine (g/L) KCCM11137P 7.8 KCCM11137PlysCP1-1′pta-ackA 7.7 KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE 8.9KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE 9.4 lysCP1-1′pta-ackA

As shown in Table 10, it was confirmed that when the KCCM11137P strainwas enhanced with the pta-ackA activity, the amount of ornithineproduction was not increased, whereas when the KCCM11137P Tn:lysCP1-argATn:lysCP1-argE strain, which is the modified strain of Corynebacteriumglutamicum simultaneously introduced with E. coli-derived argA gene andE. coli-derived argE gene, the amount of ornithine production wasincreased by 20.5% compared to that of the KCCM11137P strain, and alsoincreased by 5.6% compared to the KCCM11137P Tn:lysCP1-argATn:lysCP1-argE strain.

Example 7: Introduction of E. coli-Derived Acs in a Strain Introducedwith E. coli-Derived argA and E. coli-Derived argE and Confirmation ofOrnithine-Producing Ability of the Strain

7-1. Preparation of a Strain Introduced with E. coli-Derived Acs into aTransposon Gene from KCCM11137-Based Strain Producing Ornithine

The acs was introduced into the transposon gene using the lysCP1promoter in order to confirm whether the introduction of E. coli-derivedacs into the KCCM11137P (Korean Patent No. 10-1372635) strain, which isa Corynebacterium glutamicum ATCC13032-based strain producing ornithine,can improve the ornithine-producing ability.

First, the plasmid pDZTn-lysCP1-acs prepared in Example 3-1 wasintroduced into KCCM11137P and KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argEstrains, respectively, by electroporation to obtain transformants, andthe transformants were plated on BHIS plate media (Braine heart infusion(37 g/L), sorbitol (91 g/L), and agar (2%)) containing kanamycin (25μg/mL) and X-gal (5-bromo-4-chloro-3-indolin-D-galactoside) and culturedto form colonies. Among the colonies, blue colonies were selected andthereby the transformed strains introduced with the plasmidpDZTn-lysCP1-acs were selected.

The selected strains were cultured with shaking (30° C., 8 hours) in CMmedia (glucose (10 g/L), polypeptone (10 g/L), yeast extract (5 g/L),beef extract (5 g/L), NaCl (2.5 g/L), urea (2 g/L), pH 6.8) andsequentially diluted from 10⁻⁴ to 10⁻¹⁰, plated on solid mediacontaining X-gal, and cultured to form colonies. Among the thus-formedcolonies, white colonies which appeared at a relatively low rate wereselected and the strain introduced with acs-encoding gene by a secondarycrossover was finally selected.

The finally selected strains were subjected to PCR using a primer pairof SEQ ID NOS: 13 and 25 and confirmed that the acs-encoding gene wasintroduced. The thus-selected modified strains of Corynebacteriumglutamicum were named as KCCM11137P Tn:lysCP1-acs and KCCM11137PTn:lysCP1-argA Tn:lysCP1-argE Tn:lysCP1-acs, respectively.

7-2. Evaluation of Ornithine-Producing Ability of a Strain Introducedwith E. coli-Derived Acs

In order to confirm the effect of the introduction of acs on a strainproducing ornithine introduced with E. coli-derived argA and E.coli-derived argE, the ornithine-producing ability was compared amongthe modified strains of Corynebacterium glutamicum prepared in Example7-1.

Specifically, two different kinds of modified strains of Corynebacteriumglutamicum, (KCCM11137P Tn:lysCP1-acs; KCCM11137P Tn:lysCP1-argATn:lysCP1-argE Tn:lysCP1-acs) and two different kinds of parent strains(KCCM11137P; KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE) were respectivelyplated on 1 mM arginine-containing CM plate media (1% glucose, 1%polypeptone, 0.5% yeast extract, 0.5% beef extract, 0.25% NaCl, 0.2%urea, 100 μL of 50% NaOH, 2% agar, pH 6.8, based on 1 L), and culturedat 30° C. for 24 hours. Each of the strains cultured therefrom in anamount of about one platinum loop was inoculated into 25 mL of titermedia (8% glucose, 0.25% soybean protein, 0.50% corn steep solids, 4%(NH₄)₂SO₄, 0.1% KH₂PO₄, 0.05% MgSO₄.7H₂O, 0.15% urea, biotin (100 μg),thiamine HCl (3 mg), calcium-pantothenic acid (3 mg), nicotinamide (3mg), 5% CaCO₃, based on 1 L), and cultured with shaking at 30° C. at arate of 200 rpm for 98 hours. In all cultures of the strains, 1 mMarginine was added to the media. Upon completion of culture, theconcentration of ornithine produced in each culture broth was measuredand the results are shown in Table 11 below.

TABLE 11 Strains Ornithine (g/L) KCCM11137P 7.8 KCCM11137P Tn:lysCP1-acs7.8 KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE 8.9 KCCM11137PTn:lysCP1-argA Tn:lysCP1-argE 9.2 Tn:lysCP1-acs

As shown in Table 11, it was confirmed that when the KCCM11137P strainwas introduced with acs, the amount of ornithine production was notincreased, whereas when the KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argEstrain, which is the modified strain of Corynebacterium glutamicumsimultaneously introduced with E. coli-derived argA gene and E.coli-derived argE gene, the amount of ornithine production was increasedby 17.9% compared to that of the KCCM11137P strain, and also increasedby 3.4% compared to the KCCM11137P Tn:lysCP1-argA Tn:lysCP1-argE strain.

Summarizing the foregoing, it was confirmed that the introduction of E.coli-derived argA and E. coli-derived argE into a strain ofCorynebacterium can increase the amount of putrescine- and ornithineproduction, and additionally, it was confirmed that the enhancement ofthe activity of pta-ackA gene within a strain of Corynebacterium or theintroduction of E. coli-derived acs can further increase the amount ofputrescine- and ornithine production.

From the foregoing, a skilled person in the art to which the presentinvention pertains will be able to understand that the present inventionmay be embodied in other specific forms without modifying the technicalconcepts or essential characteristics of the present invention. In thisregard, the exemplary embodiments disclosed herein are only forillustrative purposes and should not be construed as limiting the scopeof the present invention. On the contrary, the present invention isintended to cover not only the exemplary embodiments but also variousalternatives, modifications, equivalents and other embodiments that maybe included within the spirit and scope of the present invention asdefined by the appended claims.

The invention claimed is:
 1. A method for producing putrescine,comprising: (i) culturing a modified microorganism of the genusCorynebacterium producing putrescine in a medium, wherein activities ofN-acetylglutamate synthase from E. coli and acetylornithine deacetylasefrom E. coli are introduced into the microorganism; and (ii) recoveringputrescine from the cultured microorganism or the medium.
 2. The methodaccording to claim 1, wherein the microorganism of the genusCorynebacterium is Corynebacterium glutamicum.
 3. The method accordingto claim 1, wherein the N-acetylglutamate synthase from E. coli consistsof the amino acid sequence of SEQ ID NO:
 1. 4. The method according toclaim 1, wherein (a) the activity of phosphotransacetylase and acetatekinase operon (pta-ackA operon); (b) the activity of at least oneselected from the group consisting of acetyl gamma glutamyl phosphatereductase (ArgC), acetylglutamate synthase/ornithine acetyltransferase(ArgJ), acetylglutamate kinase (ArgB), and acetyl ornithineaminotransferase (ArgD); or (c) the activity of putrescine exporter isfurther enhanced compared to its endogenous activity.
 5. The methodaccording to claim 1, wherein the activity of acetyl-CoA synthetase(acs) from E. coli, and/or the activity of ornithine decarboxylase (ODC)is further introduced.
 6. The method according to claim 1, wherein (a)the activity of i) ornithine carbamoyltransferase (ArgF), ii) glutamateexporter, or iii) ornithine carbamoyltransferase and glutamate exporterand/or (b) the activity of acetyltransferase is further weakenedcompared to its endogenous activity.